Many leukemias, including acute myelogenous leukemia (AML), are caused by oncogenic mutations that (1) alter differentiation and (2) promote proliferation of multipotent hematopoietic progenitor cells. Most chemotherapies target proliferation of the bulk leukemia cells, but few target the differentiation defects of multipotent leukemia stem cells. Development of therapies that target leukemia stem cell differentiation has been hindered by the difficulties of modeling oncogene-disrupted cell fate decisions in vitro and the difficulties of conducting high-throughput small molecule screens in vivo. A system that models the effects of oncogenes on cell differentiation in vivo while also enabling high-throughput chemical screening could open the door to therapies that target leukemia stem cell differentiation. The oncogene AML1-ETO (AE) causes a differentiation defect in hematopoietic progenitor cells that leads to accumulation of granulocytic blast cells in AML patients. Expression of AE in developing zebrafish also disrupts hematopoietic progenitor cell differentiation, leading to accumulation of granulocytic blast cells resembling those from human AML patients. A pilot screen of 2100 small molecules conducted with AE- expressing zebrafish has identified three compound classes that block the effects of AE on hematopoietic progenitor cell differentiation. A larger small molecule screen is now proposed, with the expectation that it will discover additional compounds that antagonize AE function. Compounds discovered will be subjected to mechanistic studies and evaluated for therapeutic potential using human cells and mouse models of AML. The following specific aims are proposed: Aim 1. Screening for additional AE antagonists. Aim 2. Elucidating the mechanisms by which the AE antagonists suppress AE function. Aim 3. Validating the therapeutic utility of the AE antagonists. By exploiting the unique capabilities of the zebrafish for disease modeling and small molecule screening, the proposed project will provide powerful tools for dissecting the oncogenic pathways leading to AML and may point to promising new chemotherapeutic approaches for treating the disease.